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MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells [RNA-Seq]

ABSTRACT: RNAi-related silencing mechanisms concern as diverse biological processes as gene regulation, mostly via the miRNA pathway, and defense against molecular parasites, mainly controlled by the siRNA and the piRNA pathways. In Drosophila somatic ovarian cells, transposable elements (TEs) are repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs). We show here that a functional miRNA pathway is required for this piRNA-mediated TE transcriptional silencing to operate in this tissue. A general miRNA depletion, caused by tissue- and stage-specific knock-down of either drosha (involved in miRNA biogenesis) or AG01 and gawky (both responsible for miRNA activity) resulted in chromatin-mediated TE transcriptional desilencing and piRNA loss. For unknown reasons, the amount of piRNA produced by the traffic jam 3' UTR was apparently unaffected in miRNA-defective somatic ovarian cells. Although weaker, similar phenotypes could also be observed upon individual titration (by expression of the complementary miR-sponge) of at least three miRNAs, miR-14, miR-34 and miR-989. This work adds the maintenance of genome stability, via the piRNA-mediated TE repression, to the list of the already reported miRNA-controlled biological functions. Study of small regulatory RNA populations present in ovaries expressing either wild-type Drosha protein or the trans-dominant negative Drosha protein coupled or not with the Tub-GAL80[ts]GAL4 inhibitor.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: SEVERINE CHAMBEYRON

PROVIDER: E-GEOD-60972 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells [RIP-Seq]

Project description:RNAi-related silencing mechanisms concern as diverse biological processes as gene regulation, mostly via the miRNA pathway, and defense against molecular parasites, mainly controlled by the siRNA and the piRNA pathways. In Drosophila somatic ovarian cells, transposable elements (TEs) are repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs). We show here that a functional miRNA pathway is required for this piRNA-mediated TE transcriptional silencing to operate in this tissue. A general miRNA depletion, caused by tissue- and stage-specific knock-down of either drosha (involved in miRNA biogenesis) or AG01 and gawky (both responsible for miRNA activity) resulted in chromatin-mediated TE transcriptional desilencing and piRNA loss. For unknown reasons, the amount of piRNA produced by the traffic jam 3' UTR was apparently unaffected in miRNA-defective somatic ovarian cells. Although weaker, similar phenotypes could also be observed upon individual titration (by expression of the complementary miR-sponge) of at least three miRNAs, miR-14, miR-34 and miR-989. This work adds the maintenance of genome stability, via the piRNA-mediated TE repression, to the list of the already reported miRNA-controlled biological functions. Analyses of RNA present in 3 different genetic backgounds with or without Drosha-IP: a control, a second one expressing the wild-type Drosha protein and a third one expressing the trans-dominant negative Drosha protein in the somatic cells.

2015-04-08 | E-GEOD-60973 | biostudies-arrayexpress

Maternally deposited germline piRNAs silence the tirant retrotransposon in somatic cells

Project description:Transposable elements (TEs), whose propagation can result in severe damage to the host genome, are silenced in the animal gonad by Piwi-interacting RNAs (piRNAs). piRNAs produced in the ovaries are deposited in the embryonic germline and initiate TE repression in the germline progeny. Whether the maternally transmitted piRNAs play a role in the silencing of somatic TEs is, however, unknown. Here we show that maternally transmitted piRNAs from the tirant retrotransposon in Drosophila are required for the somatic silencing of the TE and correlate with an increase in histone H3K9 trimethylation an active tirant copy. Comparison of tirant piRNAs in two Drosophila simulans natural populations.

2013-04-09 | E-GEOD-40592 | biostudies-arrayexpress

A user-friendly chromatographic method to purify small regulatory RNAs

Project description:The discovery of the small regulatory RNA populations has changed our vision of cellular regulations. Indeed, loaded on Argonaute proteins they formed ribonucleoprotein complexes that target complementary sequences and achieved widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation form cells and/or tissues remains a crucial stage to generate robust and relevant sequencing data. In 2006, a novel strategy based on anion-exchange chromatography has been purposed as an alternative to the standard size-isolation purification procedure. However, the eventual biases of such a method have been poorly investigated. Moreover, this strategy not only relies on advanced technical skills and expensive material but is time consuming and requires an elevated starting biological material amount. Using bioinformatic comparative analysis of six independent small RNA-sequencing libraries of Drosophila ovaries, we here demonstrate that anion-exchange chromatography purification prior to small RNA extraction unbiasedly enriches datasets in bona fide reads (small regulatory RNA reads) and depletes endogenous contaminants (ribosomal RNAs and degradation products). The resulting increase of sequencing depth provides a major benefit to study rare populations. We then developed a fast and basic manual procedure to purify loaded small non coding RNAs using anion-exchange chromatography at the bench. We validated the efficiency of this new method and used this strategy to purify small RNAs from various tissues and organisms. We moreover determined that our manual purification increases the output of the previously described anion-exchange chromatography procedure. Comparison of small regulatory RNA populations obtained after three different small RNA purification procedures

2013-06-13 | E-GEOD-40748 | biostudies-arrayexpress

piRNA-mediated transgenerational inheritance of an acquired trait

Project description:The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements. Comparison of Drosophila small RNA populations in ovaries and/or eggs from 3-day-old or 25-day-old females.

2012-04-13 | E-GEOD-34506 | biostudies-arrayexpress

MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-04-08 | E-GEOD-60974 | biostudies-arrayexpress

MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells [RNA-Seq]

2015-04-08 | GSE60972 | GEO

MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells [RIP-Seq]

2015-04-08 | GSE60973 | GEO

Small RNA profiling of RNase III enzyme deficient DN3 thymocytes, Tregs, activated CD4+ T cells, and embryonic fibroblasts

Project description:Here we analyze the small RNA species in the following: 1. DN3 thymocytes following inactivation of LoxP flanked Drosha and Dicer alleles with Lck-cre 2. Tregs following inactivation of LoxP flanked Drosha and Dicer alleles with CD4-cre 3. activated CD4+ T cells following inactivation of LoxP flanked Drosha and Dicer alleles with CD4-cre 4. MEFs following inactivation of LoxP flanked Drosha and Dicer alleles with Rosa26-CreER and 4-OH tamoxifen treatment Fractionation of small RNAs (19-30nt) and cloning of only RNA with 5' phosphates and 3' hydroxyls

2010-08-18 | E-GEOD-22760 | biostudies-arrayexpress

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3â² end, yielding miRNAs from the 5â² strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. Two independent sequencing experiments (set 1 and set 2, respectively) were performed using 9 samples.

2016-03-17 | E-GEOD-77989 | biostudies-arrayexpress

Euchromatic transposon insertions trigger production of novel pi- and endo-siRNAs at the target sites in the Drosophila germline

Project description:The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA-mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3' untranslated region of actively transcribed genes induces piRNA production towards the 3'-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline. The fractions of small RNAs (19-29 nt) from ovaries of y[1]; cn[1] bw[1] sp[1] line of Drosophila melanogaster were sequenced using Illumina HiSeq 2000.

2014-02-07 | E-GEOD-46105 | biostudies-arrayexpress

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